1. Field of the Invention
The present invention relates to the field of imaging using organic molecules that bind specifically to active (but not inactive) proteases and also to compounds that change fluorescence upon binding.
2. Related Art
Background
Proteases play fundamental roles in the control of both normal and disease processes. Alterations in protease expression and activity patterns underlie many human pathological processes including cancer, arthritis, osteoporosis, atherosclerosis, and neurodegenerative disorders (Ref. 1). Thus, a detailed understanding of how, when and where a particular protease functions in a complex cellular environment is required to better understand its role in the promotion of disease. Perhaps the most powerful way to address these issues is to develop methods that allow dynamic imaging of protease activity within a living cell or organism. Protease activity is tightly regulated in both normal and disease conditions. Therefore, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism.
Recently, a number of elegant methods have been developed to image enzymatic activities using both invasive and whole body imaging methods (for review see Ref. 2). Virtually all of these methods make use of reporter substrates that when processed by a given enzyme target produce a signal that can be visualized using common imaging modalities. While these methods have clearly paved the way for the application of activity based reporters to diagnostic medicine, they suffer from several features such as lack of specificity and cell permeability, as well as rapid diffusion that may limit their use in high-resolution studies of enzyme regulation and localization.
Activity based probes (ABPs) are small molecules that modify a defined set of enzyme targets based on their ability to form specific covalent bonds with key catalytic residues (for reviews see Refs. 3-7). Since this labeling reaction is mechanism-based and requires enzyme activity, extent of probe modification serves as an indirect readout of activity levels within a given sample. Probes can be designed to target a number of different classes of enzymes through optimization of both reactive functional groups and the scaffolds used to carry the reporter tag. In the past five years, a number of new classes of ABPs have been developed and used to dissect the function of various enzyme families (see reviews). The most well-established and heavily used probes are those that target proteolytic enzymes (Refs. 1-19). ABPs that target serine and cysteine proteases have been applied to studies of protease function in processes such as parasite invasion (Ref. 20), prohormone processing (Ref. 21), transcriptional regulation (Ref. 22), cataract formation (Ref. 23), natural killer cell function (Ref. 24), and cancer progression (Refs. 25-27).
A number of ABPs carrying a range of fluorescent reporters have also been described (Refs. 8, 12, 15, 27, 28). The fluorescent group serves as a highly sensitive tag that enables visualization of labeled targets after their biochemical separation. Fluorescently labeled ABPs have also been used to directly image enzyme activity using microscopy techniques. We recently demonstrated that a fluorescent ABP can be used in a mouse model for pancreatic cancer to image cysteine protease activity during multiple stages of tumor formation (Ref. 27). The use of ABPs for imaging applications has the major advantage of the formation of a permanent covalent bond with the enzyme, thus allowing direct biochemical analysis of targets. However, the major limitation of these probes is their general fluorescence both when bound to an enzyme target and when free in solution. To overcome this limitation there are disclosed here quenched probes (qABPs) that become fluorescent only after covalent modification of a protease target.